Blocking sporulation by inhibiting SpoIIE

ABSTRACT

We have shown that the control of solventogenesis and sporulation can be genetically uncoupled in  C. acetobutylicum . In strain 824(pASspo), the absence of SpoIIE causes sporulation to be blocked at stage II. The cell remains in a vegetative state, and this allows solvent production to proceed for longer and for solvents to accumulate more rapidly and to a higher concentration. The characteristic drop in OD600 observed in wild type and control strains of  C. acetobutylicum  after 48-72 hours as the cells transition from the solventogenic phase to sporulation is notably absent in the fermentations of 824(pASspo). Mutant S (wild type background, spoIIE disrupted), Mutant BS (Mutant B background, spoIIE disrupted), Mutant HS (Mutant H background, spoIIE disrupted) and Mutant bukS (buk- background, spoIIE disrupted) were generated to create stable solvent producing bacteria with complete inactivation of the SpoIIE protein. Similarity between the SpoIIE protein of  C. acetobutylicum, B. subtilis , and other  Clostridial  species indicates that the techniques used in  C. acetobutylicum  can be applied to other solvent producing  Clostridia.

PRIOR RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/173,542 filed Jul. 1, 2005, now U.S. Pat. No. 7,432,090, whichclaims the benefit under 35 USC §119(e) to U.S. Provisional ApplicationSer. No. 60/584,727 filed Jul. 1, 2004, entitled “Blocking Sporulationby Inhibiting SPOIIE,” which is incorporated herein in its entirety.

FEDERALLY SPONSORED RESEARCH STATEMENT

This invention was made with Government support under Grant No.BES-0001288 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

REFERENCE TO MICROFICHE APPENDIX

Not applicable.

REFERENCE TO A SEQUENCE LISTING

A “Sequence Listing” with sequences labeled SEQ ID NO: 1-25 is attachedhereto. A compact disc containing a Computer Readable Form (CRF) labeled“SEQUENCE LISTING.txt” is incorporated by reference. The copy in CRF isidentical to the paper copy of the “Sequence Listing” submittedherewith.

FIELD OF THE INVENTION

The invention relates to the production of organic solvents inClostridium acetobutylicum. Decreasing activity of the Stage IISporulation Protein E (SpoIIE) increases solventogenesis in Clostridiaby inhibiting sporulation without interfering with solvent production.

BACKGROUND OF THE INVENTION

The Gram-positive, obligate anaerobe C. acetobutylicum was used for theindustrial production of the solvents acetone and butanol for over 60years in the 20th century. With chemical synthesis of acetone andbutanol proving significantly more economic, there are no industrialfermentation plants of C. acetobutylicum operational in the world today(11). However, over the last 20 years the genetics and biochemistry ofC. acetobutylicum have been investigated in detail as we try tounderstand and improve upon the processes that control the production ofsolvents. Biological sources of organic solvents will become moreeconomical as raw materials become more scarce or expensive and the needfor renewable solvent sources increase.

Whereas much is known about the biochemistry of C. acetobutylicummetabolism and the genes and proteins that catalyze these processes,relatively little is known about the genetic control of the expressionof these genes. Stage 0 Sporulation Protein A (Spo0A) controls both theonset of solventogenesis and the process of sporulation in C.beijerinckii and C. acetobutylicum (30, 20). In strain SKO1 of C.acetobutylicum, where spo0A is deleted, acetone and butanol productionis reduced to 2% and 8% of wild type levels respectively. Furthermore,SKO1 cells fail to sporulate and form extended filaments of conjoinedrods (20).

Studies have also shown that there are a considerable number of Bacillussubtilis homologues in C. acetobutylicum including sigma factors andother proteins required for sporulation (32, 28). Althoughsolventogenesis does not occur in B. subtilis, it appears that a cascadeof sigma factors and stages similar to those involved in B. subtilissporulation are present in C. acetobutylicum.

The control of solventogenesis in C. acetobutylicum is geneticallylinked to the control of sporulation, as shown by the spo0A studies (30,20). It has been suggested that solventogenesis and sporulation may begenetically uncoupled at some point during early sporulation (19),although as yet there are no reports of any attempts to do so. Ifsolventogenesis could be genetically separated from sporulation, thiswould serve as an interesting and important illustration of thecomplexity of bacterial genetic control. Additionally, it may proveuseful in bioengineering solvent producing strains of Clostridium foruse in industry. A strain of C. acetobutylicum that underwentsolventogenesis without entering sporulation would increase solventproduction without inactivation, an ideal situation for large scalecontinuous fermentations.

SUMMARY OF THE INVENTION

Clostridium strains transformed with an antisense expression vectorincreased ethanol, acetone and butanol production by 225%, 43% and 110%respectively compared to the control strains. An antisense RNA vectortargeted against spoIIE, designated pASspo was constructed and evaluatedin various C. acetobutylicum strains. The genomic spoIIE gene wasdisrupted in C. acetobutylicum strains to generate Mutant S, Mutant BS,Mutant HS, and Mutant S buk-. These strains enable the stable productionof solvents for continuous fermentation. Based on these experiments, amethod of increasing solvent production was developed wherein a decreasein Clostridial SpoIIE activity inhibits sporulation while allowingcontinued solventogenesis, thus improving solvent yield.

As used herein Stage II Sporulation Protein E (SpoIIE) is used to referto the spoIIE gene and SPOIIE gene product.

The term “isolated,” as used herein, refers to a nucleic acid orpolypeptide removed from its native environment. An example of anisolated protein is a protein bound by a polyclonal antibody, rinsed toremove cellular debris, and utilized without further processing.Salt-cut protein preparations, size-fractionated preparations,affinity-absorbed preparations, recombinant genes, recombinant protein,cell extracts from host cells that expressed the recombinant nucleicacid, media into which the recombinant protein has been secreted, andthe like are also included. The term “isolated” is used because, forexample, a protein bound to a solid support via another protein is atmost 50% pure, yet isolated proteins are commonly and reliably used inthe art.

The term “substantially purified,” as used herein, refers to nucleicacid or protein sequences that are removed from their naturalenvironment and are at least 75% pure. Preferably, at least 80, 85, or90% purity is attained.

“Purified,” as used herein refers to nucleic acids or polypeptidesseparated from their natural environment so that they are at least 95%of total nucleic acid or polypeptide in a given sample. Protein purityis assessed herein by SDS-PAGE and silver staining. Nucleic acid purityis assessed by agarose gel electrophoresis and EtBr staining.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refers to polynucleotides, which may be cDNA or RNA and which may besingle-stranded or double-stranded. The term also includes peptidenucleic acid (PNA), or to any chemically DNA-like or RNA-like material.“cDNA” refers to copy DNA made from mRNA that is naturally occurring ina cell. Combinations of the same are also possible (i.e., a recombinantnucleic acid that is part gDNA and part cDNA).

The term “oligonucleotide,” as used herein, refers to a nucleic acidsequence of at least about 15 nucleotides to 100 nucleotides, and allintegers between. Preferably, oligonucleotides are about 21 to 81nucleotides, and most preferably about 51 to 78 nucleotides. Generally,an oligonucleotide must be greater than 21 to 27 nucleotides long forspecificity, although shorter oligonucleotides will suffice in certainapplications.

The term “antisense,” as used herein, is a nucleic acid sequencecomplementary to a segment of genetic material (as mRNA) and serving toinhibit gene function. Antisense oligonucleotides can inhibit eithertranscription or translation and can be synthesized to includenon-natural nucleotides. Furthermore, an antisense oligonucleotide canbe recombinantly incorporated into a genomic, viral, or plasmid DNA andoperably linked to a promoter for expression of the antisenseoligonucleotide in vivo.

The terms “operably associated” or “operably linked,” as used herein,refer to functionally coupled nucleic acid sequences.

The terms “disruption” and “disruption strains,” as used herein, referto cell strains in which the native gene is mutated, deleted, orinterrupted in such a way as to decrease the activity of the protein.

“Reduced activity” of the SpoIIE protein is defined herein to be thatreduction sufficient to inhibit sporulation. In a preferred embodiment,the reduction in activity is at least 75% as compared with controlbacteria. Preferably, at least 80, 85, or 90% reduction in activity isattained. Use of a frame shift mutation, early stop codon, pointmutations of critical residues, or deletions or insertions cancompletely inactivate (100%) gene product by completely preventingtranscription and/or translation of active protein.

Alignments were performed using BLAST homology alignment as described byTatusova & Madden (37) and available online atwww.ncbi.nlm.nih.gov/BLAST/. The default parameters were used, exceptthe filters were turned OFF. As of Jan. 1, 2061 the default parameterswere as follows: BLASTN or BLASTP as appropriate; Matrix=none forBLASTN, BLOSUM62 for BLASTP; G Cost to open gap default=5 fornucleotides, 11 for proteins; E Cost to extend gap [Integer] default=2for nucleotides, 1 for proteins; q Penalty for nucleotide mismatch[Integer] default=−3; r reward for nucleotide match [Integer] default=1;e expect value [Real] default=10; W wordsize [Integer] default=11 fornucleotides, 3 for proteins; y Dropoff (X) for blast extensions in bits(default if zero) default=20 for blastn, 7 for other programs; X dropoffvalue for gapped alignment (in bits) 30 for blastn, 15 for otherprograms; Z final X dropoff value for gapped alignment (in bits) 50 forblastn, 25 for other programs.

Abbreviations: Ap^(R), ampicillin resistance cassette; cat, promoterlesschloramphenicol acetyl transferase gene; catP, chloramphenicol acetyltransferase open reading frame with complete promoter; Thi^(R)/Cm^(R),thiamphenicol/chloramphenicol resistance cassette with functional catP;MLS^(R), macrolide, lincosamide and streptogramin A resistance cassette;Str^(R), streptomycin resistance cassette; Ap, ampicillin; Em,erythromycin; Km, kanamycin; Cm, chloramphenicol; ColE1, Gram-negativeorigin of replication; OriII, repL, Gram-positive origin of replication;lacZ, promoterless β-galactosidase gene derived fromThermoanaerobacterium thermosulfurogenes EM1 (38); lacZ′, truncated,non-functional copy of lacZ; mcrA, ΔmcrBC, methylcytosine-specificrestriction system abolished; recAl, homologous recombination abolished;spo0A⁻, deletion of spo0A. Common restriction enzymes and restrictionsites can be found at NEB® (NEW ENGLAND BIOLABS®, www.neb.com) andINVITROGEN® (www.invitrogen.com). ATCC®, AMERICAN TYPE CULTURECOLLECTION™ (www.atcc.org).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Antisense construct to spoIIE. The spoIIE antisense constructconsists of oligonucleotide “spoasastop” (SEQ ID NO: 22) and “spoasbtm”(SEQ ID NO: 23). The single underlined GATC forms the 5′ BamHI “sticky”end. The black shaded region includes 38 bases of the spoIIE openreading frame, followed by 16 bases of the upstream leader sequence,shaded dark gray. The bold region forms the 17 base rho-independentterminator region from an antisense RNA targeted against the glnA gene,found naturally in Clostridium saccharobutylicum NCP262 (10).

FIG. 2. CAT activity in strain 824(pCATspo) (▪) and control strain824(pCATP) (□). The solvent levels are shown for acetone (▴) and butanol(♦). Data are shown ±1 standard error. For acetone and butanol, n=9; for824(pCATP) CAT activity, n=3; for 824(pCATspo) CAT activity, n=6.

FIG. 3. β-galactosidase activity in strain 824(pTLspo) (♦) and controlstrains 824(pThilac) (⋄), SK(pThilac) (□) and SK(pTLspo) (▪). Data areshown ±1 standard error. For 824(pThilac), 824(pTLspo) and SK(pTLspo),n=4; for SK(pThilac), n=3.

FIG. 4A-F. Growth and product formation fermentations of strain824(pASspo) (−) and strain 824(pASsos) (−). The name for each profile dis shown above each graph (4A=OD600; 4B=Ethanol Production; 4C=AcetoneProduction; 4D=Acetate Production; 4E=Butanol Production; 4F=ButyrateProduction). Data are shown +/−1 standard error. For each data point,n=4.

FIG. 5. Phylogenetic tree of SpoIIE in different bacterial species.SpoIIE homologues shown are the 11 most identical to SpoIIE in C.acetobutylicum, as identified in a BLAST search of known bacterialgenomes. Phylogenetic tree was generated using the PHYLIP™ format. BothBLAST and PHYLIP™ tools are available on the BIOLOGY WORKBENCH™, locatedat workbench.sdsc.edu/ (San Diego Supercomputer Center, University ofCalifornia—San Diego, Calif.).

FIG. 6. Hydropathy plot of SpoIIE. Hydropathy plots N-terminal region ofSpoIIE from B. subtilis (gray line & X-axis scale) and C. acetobutylicum(black line & X-axis scale). Gray “▾” symbols at top of graph indicatetransmembrane domains identified in SpoIIE of B. subtilis (2).

FIG. 7. Amino acid sequence alignment of SpoIIE in B. subtilis and C.Acetobutylicum. Alignment was performed on the C-terminal amino acids ofSpoIIE in B. subtilis (GENBANK(R) Acc. # P37475) (SEQ ID NO. 26) and C.acetobutylicum (GENBANK(R) Acc. # NP 349801) (SEQ ID NO. 2) using theCLUSTALW tool of the Biology Workbench (San Diego Supercomputer Center,University of California-San Diego, Calif.). Numbers indicate amino acidposition. Background shading: black=conserved residue; darkgray=conserved property; white background=no consensus. (28). Black “−”symbols indicate the invariant D610, D628, D746, G747 and D795 in B.subtilis. Common motifs around the invariant D746, G747 and D795 areshown in boxes (9, 1).

FIG. 8. Acid/solvent production in 120 hour 824(pMspo) fermentations.Graphs for 120 hour fermentations of strains 824(pIMP1), 824(pMspoD) and824(pMspo). The data for 824(pMspo) are therefore divided into thosecultures which produced solvents, designated 824(pMspo)+, and thosewhich did not, designated 824(pMspo)0. The data is arranged to allowcomparison of growth and acid/solvent production between the 4 data setsas follows; A. Optical density (600 nm), B. Ethanol production, C.Acetone production, D. Acetate production, E. Butanol production, F.Butyrate production. All data points are shown ±1 standard error. For824(pIMP1), 824(pMspoD) and 824(pMspo)+, n=4; for 824(pMspo)0, n=3.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Methods for producing organic solvents are disclosed, one example ofwhich is reducing SpoIIE activity in a solvent producing strain ofClostridium sufficiently to inhibit sporulation, culturing said strainunder conditions suitable for solventogenesis, and purifying thesolvents from the culture media.

Also provided are recombinant solvent producing Clostridia that havereduced SpoIIE protein activity sufficient to inhibit sporulation.Activity can be reduced 75%, 80%, 85%, 90%, or 95%. In a preferredembodiment the activity is reduced to essential nil. Such recombinantClostridium can be engineered to produce antisense nucleotides toinhibit SpoIIE expression, or can be provided with SpoIIE mutationssufficient to inhibit activity. The mutations can be changes in theregulatory regions, premature stop codons, frame shift mutations, largeinsertions or deletions, or point mutations of invariant residues, butin an preferred embodiment, the mutation is a knock-out. Other methodsof inhibiting SpoIIE activity can also be used.

Also provided are antisense oligonucleotides that function to decreaseSpoIIE activity sufficiently to inhibit sporulation, without decreasingsolventogenesis. Some examples are oligonucleotides comprising SEQ IDNO: 22, 23, 24, or 25. Also provided are mutant spoIIE gene sequencesthat can be used to create knock-strains or mutations that can be usedto otherwise reduce SpoIIE activity.

Escherichia coli was grown in Luria-Bertani (LB) medium aerobically at37° C. (26) appropriately supplemented with Ap at 100 μg/ml, Em at 200μg/ml, Km at 50 μg/ml or Cm at 35 μg/ml. Strains were stored at −80° C.in medium supplemented with 50% glycerol. C. acetobutylicum strains weregrown anaerobically in Clostridial Growth Medium (CGM) at 37° C. (21)appropriately supplemented with Em/Cm at 40 μg/ml or Thi at 25 μg/ml.Strains were stored as horse-serum supplemented lyophilized stocks atroom temperature or at −80° C. in medium supplemented with 10% glycerol.For the sporulation and morphology assays, strains were grown onagar-solidified CBM supplemented with Em (40 μg/ml) anaerobically at 37°C. (29).

EXAMPLE 1 Materials and Methods

TABLE 1 Strains and Plasmids Strain or plasmid Relevant characteristicsReference ATCC # Strains C. acetobutylicum Wild type ATCC ® 824 C.acetobutylicum SKO1 spoOA⁻; MLS^(R) 20 E. coli DH10β mcrA, ΔmcrBC,recA1, Str^(R) NEB ™ C. acetobutylicum MutS ΔspoIIE, catP this study C.acetobutylicum MutB Mutant B (824 solR::pO1X) 27 C. acetobutylicum MutBSMutant B ΔspoIIE, catP this study C. acetobutylicum MutH Mutant H (824solR::pO1X) 27 C. acetobutylicum MutHS Mutant H ΔspoIIE, catP this studyC. acetobutylicum M5S degenerated ΔspoIIE, catP this study C.acetobutylicum buk- Δ butyrate kinase 15 C. acetobutylicum MutS buk- Δbutyrate kinase, ΔspoIIE, catP this study Plasmids pCATP MLS^(R), OriII,ColE1ori, catP 34 pCATspo MLS^(R), OriII, ColE1ori, catP, spoIIEpromoter 35 pSA12 MLS^(R), OriII, ColE1ori, lacZ′ 43 pSC12lacZ Cm^(R),OriII, ColE1ori, lacZ′ 43 pHT3 Ap^(R), MLS^(R), ColE1ori, repL, lacZ 38pThilac Thi^(R), OriII, ColE1ori, lacZ 35 pTLspo Thi^(R), OriII,ColE1ori, lacZ, spoIIE promoter 35 pIMP1 Ap^(R), MLS^(R), ColE1ori, repL24 pMspo Ap^(R), MLS^(R), ColE1ori, repL, spoIIE 35 pMspoD Ap^(R),MLS^(R), ColE1ori, repL, spoIIE′ 35 pSpoΔ4 MLS^(R), OriII, ColE1ori,lacZ′, spoIIE this study pSOS94 ptb promoter, Ap^(R), MLS^(R), ColE1ori,repL 38 pASsos ptb promoter, Ap^(R), MLS^(R), ColE1ori, repL 35 pASspoptb promoter, Ap^(R), MLS^(R), ColE1ori, repL 35

Plasmids were purified from E. coli using the QIAPREP™ Miniprepprotocols. DNA was purified from agarose gels using the QIAQUICK™ GelExtraction Kit, and PCR product or enzymatically-manipulated DNA waspurified using the QIAQUICK™ PCR Purification Kit (QIAGEN™ Inc.,Valencia, Calif.). Plasmids were purified from C. acetobutylicumaccording to the protocol developed by Harris (18). Genomic DNA waspurified from C. acetobutylicum using the PUREGENE™ Genomic DNAPurification Kit (GENTRA SYSTEMS™, Minneapolis, Minn.).

All commercial enzymes used in this study (Taq polymerase, restrictionendonucleases, calf intestinal phosphatases, T4 DNA ligase, Klenowfragment of DNA polymerase I) were used according to the manufacturers'recommendations.

Automated DNA sequencing was performed by LONESTAR™ automated DNAsequencing (LONESTAR LABORATORIES™ Inc., Houston, Tex., www.lslabs.com).

Prior to transformation into C. acetobutylicum, E. coli plasmid DNA wasmethylated by the phi3TI methyltransferase to prevent restriction by theClostridial endonuclease Cac824I (25). This was achieved bytransformation of the required plasmid into DH10β E. coli harboringvector pDHKM (43) carrying an active copy of the phi3TImethyltransferase gene. Electrotransformation of methylated plasmidsinto C. acetobutylicum was carried out according to a modification ofthe protocol developed by Mermelstein (24). Positive transformants wereisolated on agar-solidified CGM supplemented with the appropriateantibiotic, and transformations were confirmed by plasmid DNApurification.

TABLE 2 SEQ ID NO AND DESCRIPTION SEQ ID NO: TYPE Length Name andDescription 1 DNA 2388 nt  Wild type spoIIE cDNA [NC_003030 at 3351731 .. . 3354118] 2 Peptide 795 aa  Wild type SPOIIE protein [NP_349801] 3DNA 33 nt spoprom - spoIIE primer 4 DNA 35 nt sporev - spoIIE primer 5DNA 42 nt spofor - spoIIE primer 6 DNA 30 nt ASseq - automatedsequencing primer 7 DNA 25 nt adhEleft - adhE primer 8 DNA 25 ntadhEright - adhE primer 9 DNA 38 nt sinRfor - sinR primer 10 DNA 34 ntsinRrev - sinR primer 11 DNA 36 nt spofragUP - Upstream spoIIE primer 12DNA 35 nt spofragDS - Downstream spoIIE primer 13 DNA 30 nt catPstN -catP primer 14 DNA 30 nt catPstC - catP primer 15 DNA 42 nt spoORFfor -spoIIE ORF primer 16 DNA 35 nt spoORFrev - spoIIE ORF primer 17 DNA 24nt bukDfor - butyrate kinase primer 18 DNA 21 nt bukDrev - butyratekinase primer 19 DNA 26 nt solR453 - pO1X primer, solR primer 20 DNA 23nt Tc238 - pO1X primer 21 DNA 28 nt solR1361 - solR primer 22 DNA 77 ntspoastop - spoIIE antisense oligonucleotide 23 DNA 77 nt spoasbtm -spoIIE antisense oligonucleotide 24 DNA 54 nt spoastop′ - spoIIEantisense oligonucleotide 25 DNA 54 nt spoasbtm′ - spoIIE antisenseoligonucleotide 26 Peptide 289 aa  Bacillus subtilis SPOIIE protein froma.a. 539 to 827

All assays were conducted from single colonies of transformed C.acetobutylicum grown in closed-cap batch fermentations of 100 ml CGMsupplemented with the appropriate antibiotic at 37° C. in a FORMASCIENTIFIC™ anaerobic chamber (THERMO FORMA™, Marietta, Ohio). To allowfor differences in lag time following inoculation, zero hour (T0) wasdetermined when the culture had reached an OD600 of 0.1. Fermentationswere allowed to proceed for 120 hours.

EXAMPLE 2 Determining SPOIIE Expression Patterns

To investigate the role of SpoIIE in the control of solventogenesis andsporulation in C. acetobutylicum, initial studies focused on using thespoIIE promoter with a chloramphenicol acetyl-transferase (CAT) orβ-galactosidase (β-Gal) reporter system to elucidate SPOIIE expressionpatterns. These experiments showed that spoIIE is expressed transientlyin wild type C. acetobutylicum during mid- to late solventogenesis, butthat there is no detectable expression of spoIIE in the spo0A-deletedmutant strain, SKO1. This agrees with reports that Spo0A is required forthe transcriptional activation of spoIIE in B. subtilis (42), thatspoIIE expression may be regulated by spoA, and that reduction of SPOIIEprotein could be used to separate sporulation from solventogenesis.

A. CAT Assays

A chloramphenicol acetyl-transferase (CAT) reporter plasmid, pCATspo(35), was used to investigate expression patterns for the spoIIE gene inwild-type Clostridium. The pCATP plasmid (34) is the control without anspoIIE promoter construct. By operably linking the spoIIE promoter tothe cat reporter protein, CAT activity in C. acetobutylicum strain824(pCATspo) could be used to determine the expression patterns of thewild-type spoIIE gene and compare to the control strain 824(pCATP). FIG.2 demonstrates that CAT expression (spoIIE promoter) increases at auniform rate between 42 and 54 hours to a maximum of approximately 14units CAT/mg protein at 54 hours. Over the next 24 hours, the CATactivity returns to basal levels. Combined acetone and butanolconcentrations from all cultures show that the individual cultures werein approximately the same stage of solventogenesis. These data show thatthe spoIIE promoter is active during mid- to late solventogenesis, whichis the stage of growth where the cells are transitioning from vegetativegrowth to sporulation.

B. β-Gal Assays

A thiamphenicol-resistant lacZ reporter plasmid, pTLspo (35), was usedto assay the spoIIE promoter activity in both wild-type and SKO1 (spoA-)Clostridium. Control plasmid pThilac (35) does not contain an spoIIEpromoter. As with the CAT assay, fermentation products were assayed bygas chromatography to demonstrate that the individual cultures were inthe same stages of solventogenesis. (data not shown). In both controlstrains 824(pThilac) and SK(pThilac), β-Gal activity was less than 1.2unit/mg protein in any sample (data not shown). In strain 824(pTLspo),β-Gal activity is detectable during late solventogenesis, reaching amaximum of ˜50 units/mg protein from 66 to 78 hours growth after T0.This is approximately 12 hours later than CAT activity in 824(pCATspo).Additionally, β-Gal activity continues for over 48 hours, whereas CATactivity lasted 30 hours. These differences are a reflection of thevariability in growth of C. acetobutylicum. β-Gal activity in SK(pTLspo)is not different from that observed in the control strains, and remainsless than 1.3 units/mg protein in any sample indicating that spoIIE isnot expressed in SKO1.

These experiments indicate that Clostridial spoIIE expression occursmid- to late solventogenesis, at which time the cell are expected to betransitioning between solventogenic growth and the onset of sporulation.SpoIIE expression was not observed in SKO1 (spo0A- strain) we canconclude that, as in B. subtilis, spo0A is required for the correctexpression of spoIIE (42).

EXAMPLE 3 Product Formation with Increased SPOIIE

The SPOIIE expression vector pMspo (35) was generated to assess theeffect of additional SPOIIE expression in wild-type Clostridium. ThepMspo vector comprises the wild type spoIIE open reading frame includingpromoter. The pMspoD control plasmid (35) contains the upstream anddownstream DNA (including promoter) with the open reading frame deleted.This ensures that effects seen in pMspo containing cells are notartifacts of the non-translated DNA sequences. Both constructs weregenerated in the Am^(R)/MLS^(R) pIMP1 shuttle vector (24). C.acetobutylicum strain 824(pMspo) and the control strains 824(pIMP1) andstrain 824(pMspoD) were generated by electrotransformation as described.In cultures of 824(pIMP1), 824(pMspoD) and 824(pMspo)+, productformation does not differ significantly between the strains. Acetone andbutanol concentrations reach maximums of 25-27 mM and ˜70 mM, which aretypical for fermentations of this scale. After 120 hours, most of theacetate and butyrate has been reassimilated into acetone and butanol,leaving final concentrations of 10-12 mM acetate and less than 5 mM ofbutyrate. No ethanol, acetone or butanol is produced in cultures of824(pMspo)0. This results in the accumulation of acids, and henceacetate and butyrate levels are elevated by ˜33% and ˜400% respectively,after 120 hours growth.

TABLE 3 PRODUCT FORMATION Strain Ethanol (mM) Acetone (mM) Acetate (mM)Butanol (mM) Butyrate (mM) 824(pIMP1) 7.25 ± 0.48 24.50 ± 0.87 12.50 ±1.32 69.75 ± 3.50 5.00 ± 0.41 824(pMspoD) 7.00 ± 0.71 25.50 ± 1.94 11.75± 0.48 72.75 ± 2.93 3.50 ± 0.29 824(pMspo)+ 7.25 ± 0.95 27.00 ± 2.1210.50 ± 0.65 69.50 ± 4.01 3.25 ± 0.48 824(pMspo)0 0.00 ± 0.00  0.00 ±0.00 17.67 ± 0.88  0.00 ± 0.00 37.33 ± 2.03 

Using an α-amylase assay (35), it was determined that none of thecultures of 824(pIMP1), 824(pMspoD) and 824(pMspo)+ were degenerate, butall three cultures of 824(pMspo)0 were degenerate and had lost the pSOL1megaplasmid (7, 19, 31). This degeneration event required an intact copyof the spoIIE open reading frame, as none of the cultures of 824(pMspoD)degenerated. Thus increased levels of SPOIIE did not adversely affectsolvent production but did cause degeneration of the pSOL1 megaplasmid.

EXAMPLE 5 Product Formation with SPOIIE Antisense

The antisense vector, pASspo, targeted against spoIIE was designedaccording to the method of Desai (10). Oligonucleotides “spoastop” (SEQID NO: 22) and “spoasbtm” (SEQ ID NO: 23) were diluted to aconcentration of 0.5 μg/μl. 9 μl of the “top” and 9 μl of the “btm”oligonucleotide were mixed with 2 μl of 10× STE buffer (100 mM Tris-HCl,500 mM NaCl, 10 mM EDTA, pH 8.0), and placed in a water bath set to 94°C. The water bath was allowed to cool to room temperature overnight,during which time the oligonucleotides annealed to form the antisenseconstruct shown in FIG. 1. Vector pSOS94 (GENBANK® Acc. # AY187685) wasdigested with BamHI and SfoI, and the 5.0 kb fragment was purified. Thisfragment was treated with the Klenow fragment of DNA polymerase I andself-ligated to form the control vector pASsos. The spoIIE antisenseconstruct and the 5.0 kb fragment of pSOS94 were BamHI cohesive-endligated, treated with the Klenow fragment of DNA polymerase I andself-ligated to form vector pASspo. Correct construction of pASsos andpASspo was confirmed by automated sequencing using primer “ASseq” (SEQID NO: 6) which hybridizes to pSOS94 between 148 and 118 bases upstreamof the ptb promoter.

Growth and product formation in 120 hour fermentations of strains824(pASsos) and 824(pASspo) is shown in FIG. 4. Cultures of 824(pASspo)grew significantly better than 824(pASsos) with a maximum OD600 of ˜6.5compared to ˜4.5. Maximum acetate concentrations in both strains weresimilar at ˜40 mM after 24 hours growth. However, acetate levelsdecrease rapidly in 824(pASspo) as acetate is reassimilated intoacetone, to a minimum of ˜9 mM after 48 hours growth. Acetate productionincreases again after 48 hours, which coincides with acetoneconcentrations reaching a maximum of ˜45 mM, at which they remain forthe remainder of the fermentation. This is 50% greater than maximumacetone concentrations of ˜30 mM observed in 824(pASsos) after 120 hoursgrowth.

Butyrate production in both strains does not differ significantly, butthis is not reflected in butanol production. In the control strain,butanol production follows a typical pattern, reaching a maximum of ˜66mM after 120 hours growth. 824(pASspo) exhibits a rapid increase inbutanol production between 16 and 64 hours growth, at which timepointbutanol production remains constant for the remainder of thefermentation. A maximum butanol concentration of ˜153 mM was recorded in824(pASspo), which is a 132% increase compared to the control strain.

By decreasing SPOIIE activity using an antisense oligonucleotideexpressed from the pASspo plasmid, the Clostridia spent a greater amountof time undergoing solventogenesis, were able to reproduce to a higherdensity of cells, and inhibit sporulation. The overall effect ofreducing SPOIIE activity is a drastic increase in solvent productionfrom engineered solvent producing Clostridia.

EXAMPLE 6 Morphology and Sporulation

Strains harboring pASsos and pASspo were grown simultaneously wereobserved at 24, 48, 72 and 140 hour intervals. At 24 hours growth, bothstrains are morphologically similar with cells are visible in all stagesof division, and in neither strain are sporulating cells observed. After48 hours growth, many 824(pASspo) cells can be seen dividing, whereasmajority of 824(pASsos) cells are single rods with the occasionalsporulating cell. After 72 hours, many 824(pASsos) cells can be seen tobe sporulating, but there are still some 824(pASspo) cells that are inthe process of division. Additionally, some single cells have anabnormal morphology, such that they are elongated two- to threefoldcompared to the control. Examination of several different cultures of824(pASspo) indicated that these elongated cells are common, and thatthey were not mis-identified as vegetative cells undergoing division.After 140 hours growth, sporulating cells or free endospores dominatedthe 824(pASsos) culture, with very few vegetative cells observed. Incontrast, very few sporulating cells were observed in cultures of824(pASspo), with the majority of cells appearing to be in thevegetative growth state. Of those cells which were sporulating, therewere several types of abnormal morphology that could be observed. Theseexperiments indicate that the antisense DNA was properly expressed,decreased SPOIIE activity, and inhibited, delayed or drastically reducedthe cells ability to undergo sporulation.

EXAMPLE 7 SPOIIE-Disrupted Strains

The plasmid pSpoΔ4 was constructed from a fragment of spoIIE (bases3351731 through 3354118 of the C. acetobutylicum genome, GENBANK® Acc. #NC_(—)003030). DNA spanning from the 509th base to the 1113th base ofthe open reading frame was amplified by PCR using primers “spofragUP”(SEQ ID NO: 11; containing an EcoRI restriction site) and “spofragDS”(SEQ ID NO: 12; containing an XbaI restriction site). Vector pSA12 andthe spoIIE fragment were digested with EcoRI and XbaI, and ligated toform plasmid pPreSpoΔ4. The ˜0.9 kb catP gene was PCR amplified by fromplasmid pIMPTH (16) using primers “catPstN” (SEQ ID NO: 13) and“catPstC” (SEQ ID NO: 14) both primers contain PstI restriction sites. APstI restriction site was located in the centre of the spoIIE fragment.Vector pPreSpoΔ4 and the catP gene were digested with PstI and ligatedto form vector pSpoΔ4. Correct construction of pSpoΔ4 was confirmed bysequencing with primers “spofragUP” (SEQ ID NO: 11) and “spofragDS” (SEQID NO: 12). The plasmid pSpoΔ4 was electrotransformed into C.acetobutylicum according to standard protocol. Positive transformants ofwild type cells were selected on Em media and transformants of MutantsB, H and buk- selected on Em/Thi media. Single colonies of positivetransformants were grown up overnight in CGM supplemented with Em. 20 μlwas streaked on RCM plates, and allowed to grow for 48 hours prior toreplica plating onto fresh RCM plates. Replica plating was carried out 5times. Additionally, 20 μl from the original cultures was streaked onCGM containing Em, Thi, and Thi/Em to confirm that the pSpoΔ4 plasmidwas not lost from the Em-resistant mutant strains.

A final replica plating was performed to transfer colonies onto CGMsupplemented with Thi. 10 colonies of each strain were grown up forfurther testing. All colonies were tested by purification of the genomeand PCR amplification used to confirm both the strain background and thecorrect insertion of the spoIIE disruption cassette. Genomic DNA waspurified using PUREGENE® Genomic DNA Purification Kit from (GentraSystems, Minneapolis, Minn.). The MASTERTAQ® PCR kit (EPPENDORF®) wasused for all PCRs.

TABLE 4 PCR PRODUCT CONFIRMS DISRUPTION Primer 1: SEQ ID NO. Primer 2SEQ ID NO. Wild-type Mutant product spoORFfor 15 spoORFrev 15 2.4 kb 3.3kb spoIIE- strain spoORFfor 15 catPstN 13 Null 1.7 kb spoIIE- strainbukDfor 17 bukDrev 18 0.9 kb 5.5 buk- strain solR453 19 Tc238 20 Null2.1 kb pO1X (Mut B and Mut H) solR453 19 solR1361 21 0.9 kb 7 kb MutHNull Mut B adhEleft 7 adhEright 8 2.9 kb pSOL1 Null = degenerate sinRfor9 sinRrev 10 Genomic control

Mutants disrupted for spoIIE were identified in all background strains,and were named Mutant S (wild type background, spoIIE disrupted), MutantBS (Mutant B background, spoIIE disrupted), Mutant HS (Mutant Hbackground, spoIIE disrupted) and Mutant bukS (buk- background, spoIIEdisrupted). Additionally, one strain of Mutant S degenerated and thiswas called Mutant M5S. This strain will be characterized as compared toMutant S and also M5. C. acetobutylicum strains with disrupted SpoIIEgenes provide an ideal strain for continuous fermentation and batchproduction of solvents. It is expected that the cells harboring agenomic disruption of the spoIIE knockout will provide a more stablecell strain and provide a complete inactivation of the SPOIIE protein.It is also predicted that this will dramatically increase solventproduction using these strains.

EXAMPLE 8 SPOIIE-Homologues

The gene designated CAC3205 in C. acetobutylicum has been identified asSpoIIE (28, GENBANK® Acc. # NC_(—)003030). The SPOIIE cDNA and proteincan be found at SEQ ID NO: 1 and 2. Phylogenetic analysis of the SPOIIEprotein sequences verifies isolation of the SpoIIE sequence andidentifies SPOIIE as a target in other solvent producing Clostridia.FIG. 5 shows a phylogenetic tree of C. acetobutylicum SPOIIE and its 11closest relatives from other bacterial species. The B. subtilis SpoIIEis not the closest relative to SpoIIE in C. acetobutylicum, and asexpected, the SpoIIE protein from related Clostridium are more closelyrelated than the B. subtilis protein. Therefore methods performed usingthe C. acetobutylicum SpoIIE cDNA and protein may be used in otherrelated solvent producing Clostridia.

Although B. subtilis do not undergo solventogenesis, the hydropathyplots in FIG. 6 indicate the two Clostridia and Bacillus SPOIIE proteinsdo have some similar properties and structures. B. subtilis SPOIIEconsists of two distinct regions—an N-terminal hydrophobic region thatcrosses the membrane 10 times, and the C-terminal, cytoplasmic catalyticdomain (9). The superimposed plots for SpoIIE in B. subtilis and C.acetobutylicum indicate similar regions of hydrophobicity andhydrophilicity, suggesting that the N-terminus of SpoIIE in C.acetobutylicum forms a similar membrane 10-spanning region. TheC-terminal catalytic domain of SpoIIE in C. acetobutylicum also exhibitsconservation of critical amino acids as shown in FIG. 7. The asp-610 andasp-628 residues have been shown to be conserved throughout a range ofbacterial and eukaryotic PP2C-like phosphatases, and form a metal ionbinding pocket within the active site of human PP2C (9). The twoconserved regions surrounding the invariant asp-746, gly-747 and asp-795have also been identified in SpoIIE homologues and PP2C phosphatases inS. pombe, cow, mouse, human and A. thaliana. Mutation of these invariantresidues to alanine also causes a severe decrease in sporulationefficiency in B. subtilis (1, 33). All the invariant amino acidsconserved between Bacillus and Clostridial SpoIIE are essential and canbe used to inactivate the SPOIIE protein in any number of solventogenicClostridia.

We have shown that the control of solventogenesis and sporulation can begenetically uncoupled in C. acetobutylicum. In strain 824(pASspo), theabsence of SpoIIE causes sporulation to be blocked at stage II. The cellremains in a vegetative state, and this allows solvent production toproceed for longer and for solvents to accumulate more rapidly and to ahigher concentration. The characteristic drop in OD600 observed in wildtype and control strains of C. acetobutylicum after 48-72 hours as thecells transition from the solventogenic phase to sporulation is notablyabsent in the fermentations of 824(pASspo). Mutant S (wild typebackground, spoIIE disrupted), Mutant BS (Mutant B background, spoIIEdisrupted), Mutant HS (Mutant H background, spoIIE disrupted) and MutantbukS (buk- background, spoIIE disrupted) were generated to create stablesolvent producing bacteria with complete inactivation of the SpoIIEprotein. Similarity between the SpoIIE protein of C. acetobutylicum, B.subtilis, and other Clostridial species indicates that the techniquesused in C. acetobutylicum can be applied to other solvent producingClostridia.

All of the references cited herein are expressly incorporated byreference. References are listed again here for convenience:

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1. A recombinant solvent producing Clostridium, said Clostridium being aClostridium acetobutylicum and having a SpoIIE gene disruptionsufficient to inhibit sporulation and wherein said SpoIIE disruptionincreases the production of ethanol, acetone, and/or butanol.
 2. Thesolvent producing Clostridium strain of claim 1, wherein said disruptionis constructed from the SpoIIE gene of SEQ ID NO:
 1. 3. The solventproducing Clostridium strain of claim 1, wherein said disruption isselected from the group consisting of a mutation, deletion, andinterruption of the SpoIIE gene.
 4. The solvent producing Clostridiumstrain of claim 1, wherein said disruption is mutation of a conservedSpoIIE residue.
 5. The solvent producing Clostridium strain of claim 1,wherein said disruption is a mutation of a conserved residue selectedfrom the group consisting of asp-610, asp-628, asp-746, gly-747 andasp-795, wherein the amino acids are numbered with reference to SEQ IDNO: 26 as numbered in FIG.
 7. 6. A method for producing an organicsolvent comprising: a) culturing the solvent producing strain ofClostridium of any of claims 1 to 5 under conditions suitable forsolventogenesis in order to generate a culture media; and b) purifyingan organic solvent from said culture media.
 7. The method of claim 6,wherein said organic solvent is ethanol, acetone, butanol orcombinations thereof.